Process for recovery of riboflavin



Patented Jan. 16, 1945 PROCESS FOR RECOVERY OF RIBOFLAVIN George E.Hines, Jr., 'lerre Hante, Ind., assignor I to Commercial SolventsCorporation, Terre Haute, Ind., a'corporation of Maryland No Drawing.Application May 28, 1943, Serial No. 488,903

14 Claims.

The present invention relates to a process for recovering dissolvedriboflavin from solutions thereof. More particularly, it pertains to amethod for the recovery thereof, by chemical precipitation methods.

Riboflavin has been obtained in relatively concentrated solutions fromnumerous sources. such as liver, yeast, and whey, as well as fromcertain fermented mashes, such as, for example, the residues obtainedfrom butyl alcohol fermentations. In isolating riboflavin from thesesources, it has been customary to filter slurries of these substances inorder to obtain a clear solution from which riboflavin can beconcentrated, by an adsorbent such as fullers earth, kaolin, silica gel,etc. The riboflavin thus adsorbed is recovered from the resultingadsorbate by eluting with a suitable eluting agent.

In this connection, various materials have been recommended as elutingagents. Among such materials are aqueous aldehydesand ketones, as wellas alcoholic solutions of organic bases. However, such materials haveshown numerous disadvantages. For example, pyridine, or methanolsolutions thereof, are undesirable from the standpoint of cost and odor.Most of the eluting agents employed also possess the common disadvantageof extracting from the adsorbate relatively large quantities ofimpurities, as well as the riboflavin itself. A further disadvantage ofsuch processes is that they are not readily adapted to the large-scaleproduction of riboflavin, which is now being practiced.

In my copending application, U. S. Serial No. 435,418, filed March 19,1942, a process is disclosed whereby dissolved riboflavin can berecovered from nutrient solutions in the form of a precipitate whichcontains between about 60 and 90 per cent of a riboflavin precursor, byinoculatirig said solutions with an active culture of bacteria capableof producing a measured potential therein favorable to the procurementof conditions that cause the dissolved riboflavin to be converted intoits less soluble precursor. While highly satisfactory results can besecured by utilizing the above-mentioned process, it is not comin thecommercial production or that vitamin,"

by the addition of certain "ypes of soluble reducing agents to suchsoluti us. By the use of such reducing agents the dissolved riboflavinis converted into a less/soluble form that precipitates, and forconvenience will be hereinafter pletely free from certain disadvantages.First of all, as indicated above, the application of the aforesaidprocess is restricted to the recovery of riboflavin from nutrientsolutions, i. e., solutions capable of maintaining active growth of thebacteria utilized. A further disadvantage of the above-mentionedprocedure is the fact that it must be carried out under reasonablyaseptic conditions in order to obtain consistently good results, whichobviously require the exercise of considerable care in effecting suchoperations.

I have now discovered that dissolved riboflavin can be effectivelyrecovered from large volumes of solutions of the type commonlyencountered referred to as a "riboflavin precursor."

In carrying out the process of my invention, the solution containing theriboflavin is filtered,

if necessary, prior to any other preliminary treatment. Thereafter, thesolution is adjusted to a .pH of between about 2.0 and 7.5, preferablybetween 5.0 and 5.5, after which a soluble reducing agent having an Eless negative thanabout -0.15 volt is added thereto at a temperature ofbetween approximately 4 to 45 C., preferably, from 20 to 30 C., in anamount sufllcient to produce an En of between about 0.050 and 0.400

' volt, during the subsequent precipitation and sedimentation steps.Almost immediately thereafter, the precursor will be observedtoprecipitate. The precipitated mass thus obtained, however, is difficultto filter satisfactorily. Therefore, because of fact, it is preferableto let the mixture stand until the precipitate has substantiallycompletely settled to the bottom. When this is accomplished; thesupernatant liquid isdecanted, and the residue filtered, usually in thepresence of a suitable filter aid, such as, for example,

Filter-Cel which is a finely-divided diatomaceous earth having anaverage particle size of from about 1 1:012

Riboflavin may be recovered from the resultant residue and purified, inaccordance with any of several procedures. I prefer, however, toaccomplish such object by the method described .in cop'endingapplication U. S. Serial No. 435,481, filed March 23, 1942, by J. K.Dale. According to that procedure; the aforesaid residue is extractedwith a suitable solvent for the riboflavin precursor. This extractionstep is generally carried out for a period of from five to ten minutes,or longer, inthe presence of agitation. and at a temperature of betweenabout and C. After this treatment the insoluble materials are removed byfiltration, and the filtrate rapidly chilled to a temperature of between15"and 25 C., while air is simultaneously introduced into the chilledsolution; As a result of this operation, the precursor is converted intobright yellow crystals of ribo flavin which may be readily separatedfrom the mother liquor by filtration, or any other conven ient means.

In order to obtain consistently good results, it is generally desirableto employ the solvent for the extraction step in an amount correspondingto not in substantial excess or 35 of the volume of the originalsolution. Within the above-stated tity of solvent utilized in any giveninstant will, in part at least, be dictated by the percentage ofriboflavin precursor in the residue obtained by filtration. Ordinarilyit will be found that such residue contains from about 60 to 80 per centprecursor. Numerous materials will serve satisfactorily as extractantsin my process. Examples of such substances are water, aqueous solutionsof lower aliphatic alcohol, propylene glycol, carbitol, methyl carbitol,and the like.

The successful operation of my process is largely dependent upon themaintenance of proper reducing conditions in the solution from which itis desired to precipitate the riboflavin precursor. Inthis connection, Ihave found that for best results the measured potential of said solutionshould lie between about -0.200 and 0.650 volt. Potential substantiallymore negative than the lattgjigure tend to cause decomposition of theaforesaid precursor, and hence should be avoided. The above-mentionedvalues were determined at 22 C. by measuring the potential of a cellconsisting of a gold-plated platinum electrode and a saturated calomelelectrode, both of which were in contact with the riboflavin-containingsolution. The period of time within which the abovestated potentialrange is reached, will, in general, depend on the strength and quantityof the particular reducing agent employed, and also the pH andtemperature of the solution. Ordinarily, the desired potential isobtained within a few minutes after addition of the reducing agent. Suchpotential represents the measured potential of the system, or expressedotherwise, the potential of the cell chain. However, the potential offundamental importance in my process i that which results from. thepotential difference across the interface existing between thegold-plated platinum electrode and the cell solution, which in thepresent invention is a riboflavin-containing solution. This potential isrepresented by the symboi Eh, the relationship of such factor to themeasured potential of the cell chain being ex pressed by the equation:

Eh=Ec+Er where Ec represents the measured potential ofthe cell chain,and Er is the potential of the reference electrode, which in thepresent-case is a saturated calomel half-cell. The value for E1: at 22C., the temperature at which the potentials were measured, is +0.250volt.

The quantity of reducing agents employed in carrying out my invention,varies within a relatively wide range, and will be found to depend uponseveral factors, one of which is the measured potential of theriboflavin solution. For example, if the potential of the originalsolution is comparatively high, there will obviously be required ahigher proportion of reducing agent to bring the En down to a value thatlies within the critical range of about +0.050 and -0.400 volt. However,when the initial potential of the solution is relatively low, of course,a smalleramount of reducing agent will be needed to obtain the potentialthat falls within the aforesaid range. When the solution has beenallowed to come in contact with the atmosphere for an undue length oftime, the reducing conditions are disturbed and frequently the Eu ischanged to a value that falls outside of the aforesaid range causing theprecipitate to go back into solution. To avoid such undesirablecondition it will be frequently found necessary to make periodicadditions of reducing agent to the solution.

Generally, it will be observed that the reducing agent can be employedin amounts varying from 2 to 5 moles per mole of dissolved riboflavin.Ordinarily no benefit will be derived by using an excess of reducingagent greater than a mole ratio of 5-to l, and such may actually proveharmful since the conditions resulting from higher cor.- centrations ofreducing agent may promote decomposition of the riboflavin precursor.Also, in this connection, it may be said that solutions containingdissolved riboflavin in concentrations below about 20 g. per ml. are,for all practical purposes, inoperative in my process. It will, ofcourse, be obvious that riboflavin may be recovered, in accordance withmy invention, from solutions containing less than 20 g. per ml.,provided that prior to the use of a reducing agent, the riboflavincontent of said solution is increased to the aforesaid value byevaporation, or any other convenient means.

While optimum results are, in general, obtained at temperatures ofbetween about 20 and 30 C. and within a pH range of about 5.0 to 5.5, aspreviously indicated precipitation will occur at temperatures rangingfrom about 4 to 45 C. and at pH values of from about 2.0 to 7.5. In thisconnection, I have observed that at the lower 'pH values a less negativeEh is required to effect precipitation at a given temperature, but atany fixed pH, the Eh necessary for precipitation will be found to becomemore negative with increasing temperatures. Furthermore, at any giventemperature, the Eu required to effect precipitation becomes morenegative with increasing pH of the solution. Otherwise stated, the Ennecessary to bring about precipitation of the riboflavin precursorvaries inversely with the increment of the pH and temperature of thesolution utilized.

Materials capable of functioning as reducing agents in my process areany of those substances soluble in the riboflavin solution, and havingan E less negative than about 0.15 volt as measured at unit activity at25 C. Such materials are capable of producing an En in solutions of thetype contemplated, of between about +0.050 and -'0.400 volt. Specificexamples of reducing agents capable of producing a potential within thedesired range are compounds that furnish the following ions: 8204 Sn+Ti+++, Cr, and V. Compounds which furnish such ions and which aresoluble in riboflavin solutions of the type contemplated by myinvention, are titanium trichloride, stannous chloride, sodiumdithionite hypovanadous sulfate, hypovanadous chloride, chromous"chloride, chromous sulfate, stannous chloride, and the like.

In the examples which follow, the yields are based upon the valueobtained by dividing the weight of riboflavin initially present into thequantity isolated in, the form of crystals, plus the riboflavinremaining in the mother liquor.

The conversion figures were calculated by dividing the weight ofcrystals isolated, by the riboflavin initially present.

Example I through a Buchner funnel. The residue thus obtained was thenextracted with 2 liters of 75 per cent isopropyl alcohol, at atemperature of 80 C. The insoluble impurities were then removed byfiltration, and the filtrate rapidly chilled to a temperature of about20 C., during which time air was introduced at the bottom of thecontaining vessel. As a' result of this treatment, bright yellowcrystals of riboflavin were obtained as a precipitate. These crystalswere isolated from the mother liquor by filtration, and, when dry,amounted to 8 grams. The mother liquor, on-

assay, showed 1.67 grams of riboflavin, indicating a yield of 90.5 percent and a. conversion of 74.5 per cent. 7

Example ll One thousand five hundred and sixty grams of stannouschloride was added, at a. temperature of C. to 370 gallons of a solutionhaving a pH of 5.1 and containing riboflavin in a concentration of 311g. per ml. The resulting mixture was then permitted to stand for aperiod of 18 hours in order to allow for complete sedimentation of theriboflavin precursor, the En of the solution during this time beingabout -0.290 volt. At the conclusion of this period, the mother liquorwas decanted and the residue filtered through a 12-inch 12-plate Sperryfilter-press. The filter cake thus obtained was next extracted with 19gallons of 75 per cent isopropyl alcohol, and the dissolved riboflavincrystallized therefrom in accordance with the procedure of Example I. Atotal of 227 grams of crystalline riboflavin was thus obtained, whilethe mother liquor from the crystallization step was found to contain 159grams, corresponding to a. conversion of 43.5.per cent, and a yield of74 per cent. Example III ,1

A solution containing riboflavin in a concentration of 247 g. per ml.,and having a. pH of 5.3, was treated with an aqueous solution ofchromous sulfate at a. temperature of C., the chromous sulfate 'beingemployed in a. ratio of 5 moles for each mole of riboflavin present.After the addition of the chromous sulfate solution, the resultingmixture was allowed to stand for a period of 1 hour. At the conclusionof this time, the supernatant liquid was decanted, and the residuefiltered. The spent top liquor, on assay, showed riboflavin to 'bepresent in a. concentration of g. per ml., indicating a recovery ofriboflavin from the original solution in an amount corresponding to ayield of 87.9 per cent.

Example IV To a solution having a pH of 5.2 811C? containing riboflavinin a concentration of 1500 pg. per ml., was added, at a temperature of28 C., an aqueous solution of hypovanadous sulfate in a ratio of 5 molesof the latter to 1 mole of riboflavin. The resulting mixture was allowedto stand for 1 hour, after which the mother liquor was. decanted, andthe residue filtered. The decanted'mother liquor was found to containriboflavin in a. concentration of 350 #8. per ml., indicating a yield of70 per cent by difference.

Example V Seven and two-tenths grams of titanium trichloridewas added to14.5 liters of a solutionhaving a pH of 5.2 and containing-riboflavin ina concentration of 240 #8. per ml. The resulting mixture was allowed tostand for a period of 1'l'/ hours at a temperature of 28 C. At theconclusion of this period, the supernatant liquid wasdecanted, and thesemi-solid residue filtered.

The decanted liquor, on assay, showed riboflavin to be present in a.concentration of 36 g. per ml., indicating a recovery yield of 85 percent. The residue, obtained by filtration, was extracted with 725 ml. of75 per cent isopropylalcohol, at

80? C., in accordance with the method described in Example I. Theriboflavin thus recovered. in the form of crystals, amounted to 2.4grams, corresponding to a conversion of 69 per cent.

It will be apparent that I have made a distinct and basic discovery inthe art of recovering riboflavin in crystalline form from its solutions,since the isolation of that vitamin from solutions thereof, by chemicalprecipitation methods, was, prior to the present invention, unknown. Inview, therefore, of the basic character of my invention, it isconsidered that any method involving the recovery of crystallineriboflavin from its solutions, based upon the use of a soluble reducingagent capable of producing an Eh in such solution of between about+0.050 and 0.400

volt, lies within the scope thereof.

Now having described my invention, what I claim is:

1. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the steps whichcomprise adding to the solution a soluble reducing agent having an Ewhich is less negative than about -0.15 volt, to produce an En in saidsolution corresponding to a value of between about +0.050 and 0.400volt, filtering the resultant mixture, and isolating the crystallineriboflavin from the residue.

2. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the step whichcomprises adding thereto a soluble reducing agent having an E which isless negative than about -0.l5 volt, to produce an Eh in said solutioncorresponding to a value of between about +0.050 and 0.400 volt, saidreducing agent being employed in a ratio of from about 2 to 5 moles permole of riboflavin present.

3. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the steps whichcomprise adjusting the initial pH of the solution to a value rangingfrom about 2.0 to 7.5, and adding to the solution a soluble reducingagent having an E' which is less negative than about 0.15 volt, to

produce an En in said solution corresponding to a value of between about+0.050 and 0.400 volt, and precipitating said riboflavin in the form ofa precursor, the Eh required to eifect said precipitation varyinginversely with the increment of the pH.

4. In a. process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 ,ug. per ml., the steps whichcomprise adjusting the initial pH of the solution to.

a value ranging from about 2.0 to 7.5, and adding to the solution at a.temperature of about 4 to 45 0., a soluble reducing agent having an 12'which is less negative than about -0.15 volt,

' and Sn++.

to produce an Eh in said solution corresponding to a value of betweenabout +0.050 and -0.400 volt, and precipitating said riboflavin in theform of a precursor, the Eu required to eifect said precipitation varyininversely with the increment of the pH and temperature.

5. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the steps whichcomprise adjusting the initial pH of the solution to a value rangingfrom about 5.0to 5.5, and adding to the solution at a temperature ofabout 20 to 30 C., a soluble reducing agent having an HP which is lessnegative than about 0.15 volt,

.to produce an Eh in said solution corresponding to a value of betweenabout +0.050 and 0.400 volt, and precipitating said riboflavin in theform of a precursor, the Eh required to effect said precipitationvarying inversely with the increment of the pH and temperature.

6. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per mil., the step whichcomprises adding to the solution a soluble reducing agent capable offurnishing ions selected from the class consisting of 8:04P, V++, Cr++,Ti+++,

7. In a process for the recovery'of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the steps whichcom-- prise adjusting "the initial pH of the solution to a value rangingfrom about 2.0 to 7.5, and adding to the solution a soluble reducingagent capable of furnishing, ions selected from the class consisting ofS:O4=, V++, Cr++, Ti+++', and Sn++.

8. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per-ml., the steps whichcomprise adjusting the initial pH of the solution to a value rangingfrom about 2.0 to 7.5, and adding to the solution at a temperature ofabout 4 to 45 C., a soluble reducing agent capable of furnishing ionsselected from the class consisting of S2O4=, Cr++, V++, Ti+++, and Sn++.

9. In a process for the recovery of riboflavin from solutions havinga-riboflavin concentration of at least 20 g. per ml., the steps whichcomprise adjusting the initial pH of the solution to a value rangingfrom about 5.0 to 5.5, and adding to the solution at a temperature ofabout 20 to 30 0., a. soluble reducing agent capable of imnishing ionsselected from the class consisting of S2O4=, Cr++, V++. Ti+++ and Sn++.

10. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the steps whichcomprise adding to the solution a soluble reducing agent having an Ewhich is less negative than about 0.15 volt to produce a total potentialin said solution corresponding to a value 01' between about 0.200 and0.6502v01t, filtering the resultant mixture and isolating thecrystalline riboflavin from the residue.

11. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the steps whichcomprise adding to the solution a soluble reducing agent having an Ewhich is less negative than about 0.15 volt to produce a total potentialin said solution corresponding to a value of between about 0.200 and0.650 volt, said reducing agent being employed in a ratio of from about2 to 5 moles per mole of riboflavin present. I

12. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the step whichcomprises adding sodium dithionite to the solution.

13. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the step which com--prises adding titanium tr-ichloride to the solution.

14. In a process for the recovery of riboflavin from solutions having ariboflavin concentration of at least 20 g. per ml., the step whichcomprises adding stannous chloride to the solution.

GEORGE E. was. it.

